Gene therapy, combined with autologous hematopoietic stem cell (HSC) transplantation, has the potential to treat a variety of nonmalignant, congenital hematologic disorders. Gene transfer to HSC followed by transplantation of those HSC into fully myeloablated subjects can produce high levels of gene-corrected blood cells in vivo. Unfortunately, intensive conditioning regimens are associated with significant toxicity. As a result, submyeloablative conditioning has been proposed as an alternative transplantation method. Transplantation of transduced HSC into submyeloablated syngeneic hosts, however, is less efficient than transplantation of transduced HSC into fully ablated hosts. This laboratory recently observed that marrow cells cultured ex vivo during gene transfer acquire an engraftment defect that is evident in submyeloablated hosts. Because most transduction protocols involve ex vivo culture, this engraftment defect is likely to impede efforts to achieve clinically relevant levels of gene-corrected blood cells in the submyeloablative setting. The current understanding of the mechanisms governing HSC self-renewal and how these processes are altered during ex vivo culture are incomplete; a better understanding of both are necessary for improved clinical outcomes. The experiments proposed in this application will determine factors important for the engraftment of transduced HSC in two models of submyeloablative conditioning for murine hosts, low dose radiation and a novel antimetabolite-based regimen. We hypothesize that the manipulations required for HSC transduction produce changes in HSC function that negatively impact engraftment in submyeloablated hosts. We plan to test this hypothesis, in murine syngeneic and xenogeneic human-NOD/SCID mouse models, using the following specific aims: 1) Determine the extent to which ex vivo transduction of HSC under optimized conditions improves engraftment in submyeloablated hosts; 2) Determine the molecular mechanism by which ex vivo transduction impairs HSC engraftment in submyeloablated hosts; and 3) Determine the capacity of non-HSC populations present in the marrow to enhance long-term engraftment of transduced HSC in submyeloablated hosts. The findings from these studies, together with the technical expertise and scientific direction acquired from further mentored training during this award period, will form the foundation for future independent investigations into mechanisms responsible for maintaining human HSC self-renewal and engraftment in the setting of hematopoietic cell gene therapy.